bims-enlima 2025-02-02 papers

bims-enlima

Biomed News

on Engineered living materials

Issue of 2025–02–02
twenty-two papers selected by
Rahul Kumar, Tallinna Tehnikaülikool

PEARL: Protein Eluting Alginate with Recombinant Lactobacilli.
Engineering electrogenetic interfaces for mammalian cell control.
Machine learning for synthetic gene circuit engineering.
Multi-step functionalization of hydrogels through mechano- and photo-responsive linkages.
Xolography for Biomedical Applications: Dual-Color Light-Sheet Printing of Hydrogels With Local Control Over Shape and Stiffness.
Signs of damage that drive protein degradation.
On-Demand Photoactivation of DNA-Based Motor Motion.
Measuring XNA polymerase fidelity in a hydrogel particle format.
Chemical and temporal manipulation of early steps in protein assembly tunes the structure and intermolecular interactions of protein-based materials.
Regio-Selective Mechanical Enhancement of Polymer-Grafted Nanoparticle Composites via Light-Mediated Crosslinking.
Genetically encoded mechano-sensors with versatile readouts and compact size.
A genetically encoded fluorescent biosensor for visualization of acetyl-CoA in live cells.
Degradable thermosets via orthogonal polymerizations of a single monomer.
Synergizing bioprinting and 3D cell culture to enhance tissue formation in printed synthetic constructs.
Time Code for multifunctional 3D printhead controls.
Dynamic flow control through active matter programming language.
Self-assembling Depsipeptides on Aggregation-Induced Emission Luminogens: A New Way to Create Programmable Nanovesicles and Soft Nanocarriers.
Logic-Gated DNA Intelligent Nanorobots for Cellular Lysosome Interference and Enhanced Therapeutics.
Cell-Free Systems to Mimic and Expand Metabolism.
Do the Shuffle: Expanding the Synthetic Biology Toolkit for Shufflon-like Recombination Systems.
Multiplexed spatial mapping of chromatin features, transcriptome and proteins in tissues.
Precision engineering of the probiotic Escherichia coli Nissle 1917 with prime editing.

Small. 2025 Jan 28. e2408316

PEARL: Protein Eluting Alginate with Recombinant Lactobacilli.

Varun Sai Tadimarri, Marc Blanch-Asensio, Ketaki Deshpande, Jonas Baumann, Carole Baumann, Rolf Müller, Sara Trujillo, Shrikrishnan Sankaran.

  Engineered living materials (ELMs) made of bacteria in hydrogels have shown considerable promise for therapeutic applications through controlled and sustained release of complex biopharmaceuticals at low costs and with reduced wastage. While most therapeutic ELMs use E. coli due to its large genetic toolbox, most live biotherapeutic bacteria in development are lactic acid bacteria due to native health benefits they offer. Among these, lactobacilli form the largest family of probiotics with therapeutic potential in almost all sites of the body with a microbiome. A major factor limiting the use of lactobacilli in ELMs is their limited genetic toolbox. This study expands on recent work to expand the genetic programmability of probiotic Lactiplantibacillus plantarum WCFS1 for protein secretion and encapsulate it in a simple, cost-effective, and biocompatible core-shell alginate bead to develop an ELM. The controlled release of recombinant proteins is demonstrated, even up to 14 days from this ELM, thereby terming it PEARL - Protein Eluting Alginate with Recombinant Lactobacilli. Notably, lactobacillus encapsulation offered benefits like bacterial containment, protein release profile stabilization, and metabolite-induced cytotoxicity prevention. These findings demonstrate the mutual benefits of combining recombinant lactobacilli with alginate for the controlled and sustained release of proteins.

Keywords:  alginate; engineered living materials; lactobacillus; secretion; synthetic biology

DOI:  https://doi.org/10.1002/smll.202408316
Cell Chem Biol. 2025 Jan 20. pii: S2451-9456(25)00003-0. [Epub ahead of print]

Engineering electrogenetic interfaces for mammalian cell control.

Maysam Mansouri, Martin Fussenegger.

  Human body cells and our daily electronic devices both communicate information within their distinct worlds by regulating the flow of electrons across specified membranes. While electronic devices depend on the flow of electrons generated by conductive materials to communicate within a digital network, biological systems use ion gradients, created in analog biochemical reactions, to trigger biological data transmission throughout multicellular systems. Electrogenetics is an emerging concept in synthetic biology in which electrons generated by digital electronic devices program customized electron-responsive biological units within living cells. In this paper, we outline endeavors to design direct electrogenetic interfaces to control cell behaviors in therapeutically engineered mammalian cells. We also discuss prospects for the world of electrogenetics, focusing on how to engineer the next generation of therapeutic cells controlled by electronic devices and the internet of the body.

Keywords:  electrogenetics; genetic circuits; mammalian cell; synthetic biology; therapeutic cell therapy

DOI:  https://doi.org/10.1016/j.chembiol.2025.01.003
Curr Opin Biotechnol. 2025 Jan 27. pii: S0958-1669(25)00007-2. [Epub ahead of print]92 103263

Machine learning for synthetic gene circuit engineering.

Sebastian Palacios, James J Collins, Domitilla Del Vecchio.

Synthetic biology leverages engineering principles to program biology with new functions for applications in medicine, energy, food, and the environment. A central aspect of synthetic biology is the creation of synthetic gene circuits - engineered biological circuits capable of performing operations, detecting signals, and regulating cellular functions. Their development involves large design spaces with intricate interactions among circuit components and the host cellular machinery. Here, we discuss the emerging role of machine learning in addressing these challenges. We articulate how machine learning may enhance synthetic gene circuit engineering, from individual components to circuit-level aspects, while highlighting associated challenges. We discuss potential hybrid approaches that combine machine learning with mechanistic modeling to leverage the advantages of data-driven models with the prescriptive ability of mechanism-based models. Machine learning and its integration with mechanistic modeling are poised to advance synthetic biology, but challenges need to be overcome for such efforts to realize their potential.

DOI: https://doi.org/10.1016/j.copbio.2025.103263
Mater Horiz. 2025 Jan 29.

Multi-step functionalization of hydrogels through mechano- and photo-responsive linkages.

Zihao Li, Chavinya D Ranaweera, Kang Lin, Yuwan Huang, Thomas G Molley, Lei Qin, Jamie J Kruzic, Kristopher A Kilian.

Patterning soft materials with cell adhesion motifs can be used to emulate the structures found in natural tissues. While patterning in tissue is driven by cellular assembly, patterning soft materials in the laboratory most often involves light-mediated chemical reactions to spatially control the presentation of cell binding sites. Here we present hydrogels that are formed with two responsive crosslinkers-an anthracene-maleimide adduct and a disulfide linkage-thereby allowing simultaneous or sequential patterning using force and UV light. Hydrogels were formed using poly(ethylene glycol)-based crosslinkers, yielding homogeneous single networks where the mechanical properties can be controlled with crosslinker content. Compression with a PDMS stamp inked with a cysteine-terminated peptide leads to (1) force-mediated retro-Diels Alder revealing a pendant maleimide and (2) subsequent Michael-type addition of the peptide. Successful functionalization was verified through monitoring anthracene fluorescence and via cell adhesion to the immobilized peptides. The material was further functionalized using UV light to open the disulfide bond in the presence of a maleimide-terminated peptide, thereby allowing a second immobilization step. Sequential derivatization was demonstrated by adding a second cell type, yielding patterns of multiple cell populations. In this way, force and light serve as complementary triggers to create geometrically structured heterotypic cell cultures for next-generation bioassays and materials for tissue engineering.

DOI: https://doi.org/10.1039/d4mh00761a
Adv Mater. 2025 Jan 27. e2410292

Xolography for Biomedical Applications: Dual-Color Light-Sheet Printing of Hydrogels With Local Control Over Shape and Stiffness.

Lena Stoecker, Gerardo Cedillo-Servin, Niklas F König, Freek V de Graaf, Marcela García-Jiménez, Sandra Hofmann, Keita Ito, Annelieke S Wentzel, Miguel Castilho.

  Current challenges in tissue engineering include creation of extracellular environments that support and interact with cells using biochemical, mechanical, and structural cues. Spatial control over these cues is currently limited due to a lack of suitable fabrication techniques. This study introduces Xolography, an emerging dual-color light-sheet volumetric printing technology, to achieve control over structural and mechanical features for hydrogel-based photoresins at micro- to macroscale while printing within minutes. A water-soluble photoswitch photoinitiator system and a library of naturally-derived, synthetic, and thermoresponsive hydrogels for Xolography are proposed. Centimeter-scale, 3D constructs with positive features of 20 µm and negative features of ≈100 µm are fabricated with control over mechanical properties (compressive moduli 0.2 kPa-6.5 MPa). Notably, switching from binary to grayscaled light projection enables spatial control over stiffness (0.2-16 kPa). As a proof of concept, grayscaled Xolography is leveraged with thermoresponsive hydrogels to introduce reversible anisotropic shape changes beyond isometric shrinkage. Xolography of viable cell aggregates is finally demonstrated, laying the foundation for cell-laden printing of dynamic, cell-instructive environments with tunable structural and mechanical cues in a fast one-step process. Overall, these innovations unlock unique possibilities of Xolography across multiple biomedical applications.

Keywords:  bioprinting; grayscale; stiffness control; thermoresponsive; volumetric 3D printing

DOI:  https://doi.org/10.1002/adma.202410292
Nature. 2025 Jan 29.

Signs of damage that drive protein degradation.

Alfred Freeberg, Michael Rapé.



Keywords:  Cell biology; Proteomics

DOI:  https://doi.org/10.1038/d41586-025-00082-7
ACS Nano. 2025 Jan 30.

On-Demand Photoactivation of DNA-Based Motor Motion.

Selma Piranej, Hiroaki Ogasawara, Luona Zhang, Krista Jackson, Alisina Bazrafshan, Khalid Salaita.

  A major challenge in the field of synthetic motors relates to mimicking the precise, on-demand motion of biological motor proteins, which mediates processes such as cargo transport, cell locomotion, and cell division. To address this challenge, we developed a system to control the motion of DNA-based synthetic motors using light. DNA motors are composed of a central chassis particle modified with DNA "legs" that hybridize to RNA "fuel", and move upon enzymatic consumption of RNA. We first concealed RNA fuel sites using photocleavable oligonucleotides that block DNA leg binding. Upon UV activation, the RNA blocking strands dissociate, exposing the RNA fuel and initiating active, directional motion. We also created a "brake" system using photocleavable DNA stalling strands, anchoring the motors until UV light removes the "brake" while simultaneously "fueling" the motors, initiating spatiotemporally controlled stop → go motion. Additionally, we modified the "brake" system to activate the motors via a chemical input, while an optical input is required to fuel the motors. This dual-input approach, functioning as an "AND" gate, demonstrates the potential for DNA motors to perform light-triggered computational tasks. Our work provides a proof of concept for enhancing the complexity and functionality of synthetic motors.

Keywords:  DNA-based motors; directional motion; molecular machines; on-demand motion; photocleavable group; synthetic motors

DOI:  https://doi.org/10.1021/acsnano.4c13068
Nucleic Acids Res. 2025 Jan 24. pii: gkaf038. [Epub ahead of print]53(3):

Measuring XNA polymerase fidelity in a hydrogel particle format.

Esau L Medina, John C Chaput.

Growth in the development of engineered polymerases for synthetic biology has led to renewed interest in assays that can measure the fidelity of polymerases that are capable of synthesizing artificial genetic polymers (XNAs). Conventional approaches require purifying the XNA intermediate of a replication cycle (DNA → XNA → DNA) by denaturing polyacrylamide gel electrophoresis, which is a slow, costly, and inefficient process that requires a large-scale transcription reaction and careful extraction of the XNA strand from the gel slice. In an effort to streamline the assay, we developed a purification-free approach in which the XNA transcription and reverse transcription steps occur inside the matrix of a hydrogel-coated magnetic particle. Accordingly, a DNA primer cross-linked throughout the gel matrix is annealed to a template of defined sequence and extended with XNA. Following removal of the DNA template, the XNA product strand is copied back into DNA, recovered, amplified, cloned, and sequenced. Performing the replication cycle in the hydrogel format drastically reduces the time and reaction scales required to measure the fidelity of an XNA polymerase, making it easier to evaluate the properties of a range of candidate XNA polymerases.

DOI: https://doi.org/10.1093/nar/gkaf038
Protein Sci. 2025 Feb;34(2): e70000

Chemical and temporal manipulation of early steps in protein assembly tunes the structure and intermolecular interactions of protein-based materials.

Valeria Italia, Amanda Jons, Bhavika Kaparthi, Britt Faulk, Marco Maccarini, Paolo Bertoncello, Ken Meissner, Donald K Martin, Sarah E Bondos.

  The Drosophila intrinsically disordered protein Ultrabithorax (Ubx) undergoes a series of phase transitions, beginning with noncovalent interactions between apparently randomly organized monomers, and evolving over time to form increasingly ordered coacervates. This assembly process ends when specific dityrosine covalent bonds lock the monomers in place, forming macroscale materials. Inspired by this hierarchical, multistep assembly process, we analyzed the impact of protein concentration, assembly time, and subphase composition on the early, noncovalent stages of Ubx assembly, which are extremely sensitive to their environment. We discovered that in low salt buffers, we can generate a new type of Ubx material from early coacervates using 5-fold less protein, and 100-fold less assembly time. Comparison of the new materials with standard Ubx fibers also revealed differences in the extent of wrinkling on the fiber surface. A new image analysis technique based on autocorrelation of scanning electron microscopy (SEM) images was developed to quantify these structural differences. These differences extend to the molecular level: new materials form more dityrosine covalent cross-links per monomer, but without requiring the specific tyrosine residues necessary for crosslinking previously established materials. We conclude that varying the assembly conditions represents a facile and inexpensive process for creating new materials. Most new biopolymers are created by changing the composition of the monomers or the method used to drive assembly. In contrast, in this study we used the same monomers and assembly approach, but altered the assembly time and chemical environment to create a new material with unique properties.

Keywords:  aggregation; biomaterials; coacervation; intrinsically disordered proteins; phase separation; protein‐based materials; self‐healing

DOI:  https://doi.org/10.1002/pro.70000
Adv Mater. 2025 Jan 28. e2410493

Regio-Selective Mechanical Enhancement of Polymer-Grafted Nanoparticle Composites via Light-Mediated Crosslinking.

Kyungtae Kim, Benjamin C Grummon, Carl J Thrasher, Robert J Macfarlane.

  Polymer-brush-grafted nanoparticles (PGNPs) that can be covalently crosslinked post-processing enable the fabrication of mechanically robust and chemically stable polymer nanocomposites with high inorganic filler content. Modifying PGNP brushes to append UV-activated crosslinkers along the polymer chains would permit a modular crosslinking strategy applicable to a diverse range of nanocomposite compositions. Further, light-activated crosslinking reactions enable spatial control of crosslink density to program intentionally inhomogeneous mechanical responses. Here, a method of synthesizing composites using UV-crosslinkable brush-coated nanoparticles (referred to as UV-XNPs) is introduced that can be applied to various monomer compositions by incorporating photoinitiators into the polymer brushes. UV crosslinking of processed UV-XNP structures can increase their tensile modulus up to 15-fold without any noticeable alteration to their appearance or shape. By using photomasks to alter UV intensity across a sample, intentionally designed inhomogeneities in crosslink density result in predetermined anisotropic shape changes under strain. This unique capability of UV-XNP materials is applied to stiffness-patterned flexible electronic substrates that prevent the delamination of rigid components under deformation. The potential of UV-XNPs as functional, soft device components is further demonstrated by wearable devices that can be modified post-fabrication to customize their performance, permitting the ability to add functionality to existing device architectures.

Keywords:  composites; nanoparticles; photocrosslinking; polymers; processing

DOI:  https://doi.org/10.1002/adma.202410493
bioRxiv. 2025 Jan 18. pii: 2025.01.16.633409. [Epub ahead of print]

Genetically encoded mechano-sensors with versatile readouts and compact size.

Yuan Ren, Jie Yang, Takumi Saito, Oliver Glomb, Sayed Iman Mousavi, Brigitte Naughton, Christina de Fontnouvelle, Barbara Fujita, Christian Schlieker, Shaul Yogev, Yongli Zhang, Julien Berro.

Mechanical forces are critical for virtually all fundamental biological processes, yet quantification of mechanical forces at the molecular scale in vivo remains challenging. Here, we present a new strategy using calibrated coiled-coils as genetically encoded, compact, tunable, and modular mechano-sensors to substantially simplify force measurement in vivo , via diverse readouts (luminescence, fluorescence and analytical biochemistry) and instrumentation readily available in biology labs. We demonstrate the broad applicability and ease-of-use of these coiled-coil mechano-sensors by measuring forces during cytokinesis (formin Cdc12) and endocytosis (epsin Ent1) in yeast, force distributions in nematode axons (β-spectrin UNC-70), and forces transmitted to the nucleus (mini-nesprin-2G) and within focal adhesions (vinculin) in mammalian cells. We report discoveries in intracellular force transmission that have been elusive to existing tools.

DOI: https://doi.org/10.1101/2025.01.16.633409
Cell Chem Biol. 2025 Jan 21. pii: S2451-9456(25)00002-9. [Epub ahead of print]

A genetically encoded fluorescent biosensor for visualization of acetyl-CoA in live cells.

Joseph J Smith, Taylor R Valentino, Austin H Ablicki, Riddhidev Banerjee, Adam R Colligan, Debra M Eckert, Gabrielle A Desjardins, Katharine L Diehl.

  Acetyl-coenzyme A is a central metabolite that participates in many cellular pathways. Evidence suggests that acetyl-CoA metabolism is highly compartmentalized in mammalian cells. Yet methods to measure acetyl-CoA in living cells are lacking. Herein, we engineered an acetyl-CoA biosensor from the bacterial protein PanZ and circularly permuted green fluorescent protein (cpGFP). The sensor, "PancACe," has a maximum change of ∼2-fold and a response range of ∼10 μM-2 mM acetyl-CoA. We demonstrated that the sensor has a greater than 7-fold selectivity over coenzyme A, butyryl-CoA, malonyl-CoA, and succinyl-CoA, and a 2.3-fold selectivity over propionyl-CoA. We expressed the sensor in E. coli and showed that it enables detection of rapid changes in acetyl-CoA levels. By localizing the sensor to either the cytoplasm, nucleus, or mitochondria in human cells, we showed that it enables subcellular detection of changes in acetyl-CoA levels, the magnitudes of which agreed with an orthogonal PicoProbe assay.

Keywords:  acetyl-CoA; biosensor; coenzyme A; metabolism; metabolite; protein engineering

DOI:  https://doi.org/10.1016/j.chembiol.2025.01.002
Nature. 2025 Jan 29.

Degradable thermosets via orthogonal polymerizations of a single monomer.

Reagan J Dreiling, Kathleen Huynh, Brett P Fors.

Crosslinked thermosets are highly durable materials, but overcoming their petrochemical origins and inability to be recycled poses a grand challenge1-3. Many strategies to access crosslinked polymers that are bioderived or degradable-by-design have been proposed, but they require several resource-intensive synthesis and purification steps and are not yet feasible alternatives to conventional consumer materials4-8. Here we present a modular, one-pot synthesis of degradable thermosets from the commercially available, biosourced monomer 2,3-dihydrofuran (DHF)9. In the presence of a ruthenium catalyst and photoacid generator, DHF undergoes slow ring-opening metathesis polymerization to give a soft polymer; then, exposure to light triggers strong acid generation and promotes the cationic polymerization of the same DHF monomer to spatially crosslink and strengthen the material10-12. By manipulating catalyst loading and light exposure, we can access materials with physical properties spanning orders of magnitude and achieve spatially resolved material domains. Importantly, the DHF-based thermosets undergo stimuli-selective degradation and can be recycled to the monomer under mild heating. The use of two distinct polymerization mechanisms on a single functional group allows the synthesis of degradable and recyclable thermoset materials with precisely controlled properties.

DOI: https://doi.org/10.1038/s41586-024-08386-w
Biofabrication. 2025 Jan 24.

Synergizing bioprinting and 3D cell culture to enhance tissue formation in printed synthetic constructs.

Daniel Günther, Cédric Bergerbit, Ary Marsee, Sitara Vedaraman, Alba Pueyo-Moliner, Céline Bastard, Guy Eelen, Jose Luis Gerardo Nava, Mieke Dewerchin, Peter Carmeliet, Rafael Kramann, Kerstin Schneeberger, Bart Spee, Laura De Laporte.

  Bioprinting is currently the most promising method to biofabricate complex tissues in vitro with the potential to transform the future of organ transplantation and drug discovery. Efforts to create such tissues are, however, almost exclusively based on animal-derived materials, like gelatin methacryloyl, which have demonstrated efficacy in bioprinting of complex tissues. While these materials are already used in clinical applications, uncertainty about their safety still remains due to their animal origin. Alternatively, synthetic bioinks are developed that match the printability of natural bioinks but lack their biological complexity, and thereby often fail to support cell growth and facilitate tissue formation. Additionally, most synthetic materials do not meet the mechanical demands to bioprint stable constructs while providing a suitable environment for cells to grow, limiting the number of available bioinks. To bridge this gap and synergize bioprinting and 3D cell culture, we developed a PEG-based bioink system to promote the growth and spreading of cell spheroids that consist of human primary endothelial cells and fibroblasts. The 3D bioprinted centimeter-scale constructs have a high shape fidelity and accelerated softening to provide sufficient space for cells to grow. Adjusting the rate of degradability, induced by the integration of ester-functionalized crosslinkers in addition to protease cleavable crosslinkers into the hydrogel network, improves the growth of spheroids in larger printed hydrogel constructs containing an interconnected channel structure. The perfusable constructs enable extensive spheroid sprouting and the formation of a cellular network upon fusion of sprouts as initial steps towards tissue formation with the potential for clinical translation.

Keywords:  Bioprinting; Hydrogels; Polyethylene glycol; Spheroids; Vascularization

DOI:  https://doi.org/10.1088/1758-5090/adae37
Nat Commun. 2025 Jan 25. 16(1): 1035

Time Code for multifunctional 3D printhead controls.

Sarah Propst, Jochen Mueller.

Direct Ink Writing, an extrusion-based 3D printing technique, has attracted growing interest due to its ability to process a broad range of materials and integrate multifunctional printheads with features such as shape-changing nozzles, in-situ curing, material switching, and material mixing. Despite these advancements, incorporating auxiliary controls into Geometry Code (G-Code), the standard programming language for these printers, remains challenging. G-Code's line-by-line execution requires auxiliary control commands to interrupt the print path motion, causing defects in the printed structure. We propose a generalizable time-based synchronization approach called Time Code (T-Code), which decouples auxiliary control from G-Code, enabling uninterrupted print path enrichment. We demonstrate the method's effectiveness with both high-end and affordable 3D printers by fabricating functional gradients and parallelizing printhead auxiliary devices for mass customization. Our method reduces defects, enhances print speed, and minimizes the mechanical burden on 3D printers, enabling the rapid creation of complex multimaterial structures.

DOI: https://doi.org/10.1038/s41467-025-56140-1
Nat Mater. 2025 Jan 29.

Dynamic flow control through active matter programming language.

Fan Yang, Shichen Liu, Heun Jin Lee, Rob Phillips, Matt Thomson.

Cells use 'active' energy-consuming motor and filament protein networks to control micrometre-scale transport and fluid flows. Biological active materials could be used in dynamically programmable devices that achieve spatial and temporal resolution that exceeds current microfluidic technologies. However, reconstituted motor-microtubule systems generate chaotic flows and cannot be directly harnessed for engineering applications. Here we develop a light-controlled programming strategy for biological active matter to construct micrometre-scale fluid flow fields for transport, separation and mixing. We circumvent nonlinear dynamic effects within the active fluids by limiting hydrodynamic interactions between contracting motor-filament networks patterned with light. Using a predictive model, we design and apply flow fields to accomplish canonical microfluidic tasks such as transporting and separating cell clusters, probing the extensional rheology of polymers and giant lipid vesicles and generating mixing flows at low Reynolds numbers. Our findings provide a framework for programming dynamic flows and demonstrate the potential of active matter systems as an engineering technology.

DOI: https://doi.org/10.1038/s41563-024-02090-w
ACS Appl Mater Interfaces. 2025 Jan 31.

Self-assembling Depsipeptides on Aggregation-Induced Emission Luminogens: A New Way to Create Programmable Nanovesicles and Soft Nanocarriers.

Carla Hernando-Muñoz, Andrea Revilla-Cuesta, Irene Abajo-Cuadrado, Camilla Andreini, Tomás Torroba, Natalia Busto, Darío Fernández, German Perdomo, Gerardo Acosta, Miriam Royo, Javier Gutierrez Reguera, Angelo Spinello, Giampaolo Barone, Dominic Black, Robert Pal.

  We introduce the proof of concept of a new methodology to produce robust hollow nanovesicles stable in water or mixtures of water and organic solvents. The bottom-up produced nanovesicles are formed by the self-assembly of depsipeptide chains of natural origin combined with new aggregation-induced emission luminogens that function as constitutional vesicle-forming moieties and fluorescent indicators of the structure of the nanovesicle. The newly formed nanovesicles are robust enough to be used to carry large molecules such as physiological peptides without losing their structural characteristics, acting as programmable nanocarrier systems within living cells as Trojan horse systems, constituting a new approach to active transport and nanoencapsulation.

Keywords:  active transport; aggregation induced emission materials; fluorescent materials; nanoencapsulation; programmed cytotoxicity

DOI:  https://doi.org/10.1021/acsami.4c19123
Angew Chem Int Ed Engl. 2025 Jan 28. e202423004

Logic-Gated DNA Intelligent Nanorobots for Cellular Lysosome Interference and Enhanced Therapeutics.

Qiwei Wang, Ruixue Chang, Xiuping Li, Ying Zhang, Xiangshan Fan, Lili Shi, Tao Li.

  Environment-recognizing DNA nanodevices have proven promising for cellular manipulation and disease treatment, whereas how to sequentially respond to different cellular microenvironments remains a challenge. To this end, here we elaborate a logic-gated intelligent DNA nanorobot (Gi-DR) for the cascade response to inter- and intra-cellular microenvironments, thereby achieving lysosome-targeted cargo delivery for subcellular interference and tumor treatment with enhanced efficacy. Utilizing G-quadruplexes to respond to high-level K+ in cancer cell surrounding, this Gi-DR nanorobot can activate an aptamer-based transmembrane DNA machine that delivers molecular payloads to cellular lysosome. Accordingly, the nanoassembly of Gi-DR is promoted by the folding of heterodimeric i-motifs in the acidic microenvironment. Such a design allows the extra-/intra-cellular behaviors of the Gi-DR nanorobot to be programmed by an YES-AND logic circuit, with environmental K+ and H+ as two inputs. As a consequence, DNA nanofibers are controllably formed in living cells, interfering with lysosomal function and thereby preventing cellular proliferation. Further, a therapeutic agent (i.e. ligand-drug conjugate) is delivered into target cancer cells for synergistic tumor treatment in vivo, exhibiting the super-enhanced cancer cell lethality and anti-tumor efficacy. It well illustrates that our designed logic-gated DNA nanorobot has broad application prospects in modulating cellular function and precision disease treatment.

Keywords:  DNA intelligent nanorobots; enhanced therapeutics; environment-recognizing; logic gate; lysosome interference

DOI:  https://doi.org/10.1002/anie.202423004
ACS Synth Biol. 2025 Jan 29.

Cell-Free Systems to Mimic and Expand Metabolism.

Blake J Rasor, Tobias J Erb.

Cell-free synthetic biology incorporates purified components and/or crude cell extracts to carry out metabolic and genetic programs. While protein synthesis has historically been the primary focus, more metabolism researchers are now turning toward cell-free systems either to prototype pathways for cellular implementation or to design new-to-nature reaction networks that incorporate environmentally relevant substrates or new energy sources. The ability to design, build, and test enzyme combinations in vitro has accelerated efforts to understand metabolic bottlenecks and engineer high-yielding pathways. However, only a small fraction of metabolic possibilities has been explored in cell-free systems, and extracts from model organisms remain the most common starting points. Expanding the scope of cell-free metabolism to include extracts from new organisms, alternative metabolic pathways, and non-natural chemistries will enhance our ability to understand and engineer bio-based chemical conversions.

DOI: https://doi.org/10.1021/acssynbio.4c00729
ACS Synth Biol. 2025 Jan 27.

Do the Shuffle: Expanding the Synthetic Biology Toolkit for Shufflon-like Recombination Systems.

Jan Katalinić, Morgan Richards, Alex Auyang, James H Millett, Manjunatha Kogenaru, Nikolai Windbichler.

  Naturally occurring DNA inversion systems play an important role in the generation of genetic variation and adaptation in prokaryotes. Shufflon invertase (SI) Rci from plasmid R64, recognizing asymmetric sfx sites, has been adopted as a tool for synthetic biology. However, the availability of a single enzyme with moderate rates of recombination has hampered the more widespread use of SIs. We identified 14 previously untested SI genes and their sfx sites in public databases. We established an assay based on single-molecule sequencing that allows the quantification of the inversion rates of these enzymes and determined cross-recognition to identify orthogonal SI/sfx pairs. We describe SI enzymes with substantially improved shuffling rates when expressed in an inducible manner in E. coli. Our findings will facilitate the use of SIs in engineering biology where synthetic shufflons enable the generation of millions of sequence variants in vivo for applications such as barcoding or experimental selection.

Keywords:  DNA inversion; Rci; barcoding; invertase; recombinase; shufflon

DOI:  https://doi.org/10.1021/acssynbio.4c00790
Nat Methods. 2025 Jan 27.

Multiplexed spatial mapping of chromatin features, transcriptome and proteins in tissues.

Pengfei Guo, Liran Mao, Yufan Chen, Chin Nien Lee, Angelysia Cardilla, Mingyao Li, Marek Bartosovic, Yanxiang Deng.

The phenotypic and functional states of cells are modulated by a complex interactive molecular hierarchy of multiple omics layers, involving the genome, epigenome, transcriptome, proteome and metabolome. Spatial omics approaches have enabled the study of these layers in tissue context but are often limited to one or two modalities, offering an incomplete view of cellular identity. Here we present spatial-Mux-seq, a multimodal spatial technology that allows simultaneous profiling of five different modalities: two histone modifications, chromatin accessibility, whole transcriptome and a panel of proteins at tissue scale and cellular level in a spatially resolved manner. We applied this technology to mouse embryos and mouse brains, generating detailed multimodal tissue maps that identified more cell types and states compared to unimodal data. This analysis uncovered spatiotemporal relationships among histone modifications, chromatin accessibility, gene expression and protein levels during neuron differentiation, and revealed a radial glia niche with spatially dynamic epigenetic signals. Collectively, the spatial multi-omics approach heralds a new era for characterizing tissue and cellular heterogeneity that single-modality studies alone could not reveal.

DOI: https://doi.org/10.1038/s41592-024-02576-0
Appl Environ Microbiol. 2025 Jan 31. e0003125

Precision engineering of the probiotic Escherichia coli Nissle 1917 with prime editing.

Pei-Ru Chen, Ying Wei, Xin Li, Hai-Yan Yu, Shu-Guang Wang, Xian-Zheng Yuan, Peng-Fei Xia.

  CRISPR-Cas systems are transforming precision medicine with engineered probiotics as next-generation diagnostics and therapeutics. To promote human health and treat disease, engineering probiotic bacteria demands maximal versatility to enable non-natural functionalities while minimizing undesired genomic interferences. Here, we present a streamlined prime editing approach tailored for probiotic Escherichia coli Nissle 1917 utilizing only essential genetic modules, including Cas9 nickase from Streptococcus pyogenes, a codon-optimized reverse transcriptase, and a prime editing guide RNA, and an optimized workflow with longer induction. As a result, we achieved all types of prime editing in every individual round of experiments with efficiencies of 25.0%, 52.0%, and 66.7% for DNA deletion, insertion, and substitution, respectively. A comprehensive evaluation of off-target effects revealed a significant reduction in unintended mutations, particularly in comparison to two different base editing methods. Leveraging the prime editing system, we inserted a unique DNA sequence to barcode the edited strain and established an antibiotic-resistance-gene-free platform to enable non-natural functionalities. Our prime editing strategy presents a CRISPR-Cas system that can be readily implemented in any laboratories with the basic CRISPR setups, paving the way for future innovations in engineered probiotics.IMPORTANCEOne ultimate goal of gene editing is to introduce designed DNA variations at specific loci in living organisms with minimal unintended interferences in the genome. Achieving this goal is especially critical for creating engineered probiotics as living diagnostics and therapeutics to promote human health and treat diseases. In this endeavor, we report a customized prime editing system for precision engineering of probiotic Escherichia coli Nissle 1917. With such a system, we developed a barcoding system for tracking engineered strains, and we built an antibiotic-resistance-gene-free platform to enable non-natural functionalities. We provide not only a powerful gene editing approach for probiotic bacteria but also new insights into the advancement of innovative CRISPR-Cas systems.

Keywords:  CRISPR; Escherichia coli Nissle 1917; prime editing; probiotics

DOI:  https://doi.org/10.1128/aem.00031-25